Cancer is one of the world's biggest killers, with estimates that 7.6 million people died of cancer in 2005—representing 13% of deaths worldwide. Between 2005 and 2015, 84 million more people will die if urgent action is not taken (WHO forecast). Such statistics have prompted a largescale investment in cancer research in both the public health and private sectors. Therefore, there is an urgent need to translate medical research towards the development of novel cancer treatments and drugs.
It was Folkman's group in the early 70's that performed definitive experiments demonstrating the release by implanted tumours of soluble factors that promote the induction of angiogenesis within the host leading to the recruitment of a blood supply supporting tumour growth and metastasis. These pioneering experiments suggested that abrogation of tumour angiogenesis would be a viable anti-cancer therapeutic strategy and this has been supported by pre-clinical studies. The development of anti-angiogenic drugs including monoclonal antibodies (Avastin) to the important angiogenic factor VEGF has led to the successful application of this strategy in the clinic. Anti-angiogenic drugs in combination with standard chemotherapy have generated impressive results in clinical trials. However, drugs such as Avastin have shown signs of promoting severe side effects and this has prompted the search for more selective and milder alternatives.
Angiogenesis is the process of neocapillary sprouting from pre-existing vessels in response to signals induced by hypoxia. Physiological angiogenesis is a finely regulated process involving the interplay between distinct vascular cell types incorporating a host of humoural regulatory molecules controlling and coordinating primarily endothelial and smooth muscle cell responses. Newly developing vessels are organised into a patterned vascular network that is directed by the hypoxic requirements of a particular organ but, nonetheless, undergo common cell-coordinated responses such as migration, proliferation, tubulogenesis, and remodelling.
The most important physiological humoural mediator of angiogenesis is VEGF-A which controls vessel permeability, endothelial cell (EC) proliferation and survival, migration and morphogenetic processes associated with vascular patterning (1). The challenge to understanding the biology of angiogenesis is the elucidation of the spatiotemporal regulation of VEGF A signalling that controls the sequential processes during capillary sprouting, growth and maturation.
Recent work has highlighted the important role of the specialisation of the endothelial compartment of sprouting vessels. Gerhardt et al showed in the retina that specialized tip cells characterised by extensive filopodia present at the migrating front of the developing vascular plexus guide vascular patterning in response to matrix associated gradients of VEGF A. In contrast, cells comprising the vessel stalk proliferate in response to soluble VEGF A concentration (2, 3). At the molecular level, we and others have shown that the extracellular matrix (ECM) component fibronectin (Fn) augments EC responses induced by VEGF A through collaborative signalling between the receptor tyrosine kinase VEGFR2 and the integrin α5β1 (4-6). We identified and mapped a VEGF binding domain within domains III12-14 of the Hep II region of Fn that augments VEGF A/VEGFR2 mediated EC responses (7). The combined activity of the Hep II VEGF binding domain and the cell-binding domain encompassing modules III9-III10 present within a single Fn fragment was indispensable for signal amplification.
While the discovery of binding domains in Fn and further VEGF A sequestration by additional ECM components such as heparan sulphate proteoglycans (2) provide molecular insights into how matrix-associated VEGF A gradients are established to drive tip cell migration, the VEGF A-dependent mechanisms that regulate capillary stalk morphogenesis and integrity are not well understood. Evidence highlighting the necessity of controlling VEGF concentration during vasculogenesis have come from studies employing VEGFR1 null mice which showed that embryonic lethality is caused by abnormal vessel development in utero characterised by vascular overgrowth, a consequence of dysregulated endothelial cell proliferation (8). Furthermore, during development, the extracellular domain of VEGFR1 was sufficient to support vasculogenesis in VEGFR1 kinase null mice supporting the notion that controlling VEGF A concentration is an important physiological parameter in regulating angiogenesis through controlling the proliferative capacity of endothelial cells (9).
These studies illustrate that during angiogenesis/vasculogenesis signalling through the VEGF A-VEGFR2 axis is regulated through multiple pathways utilising several distinct families of receptors, specially adapted endothelial subpopulations and the spatial regulation of VEGF concentration and gradients established in association with components of the ECM. This raises the possibility that additional molecules may also be involved in regulating VEGFR2 dependent processes including those regulating stalk cell behaviour. A potential candidate gene family for the regulation of VEGF signalling and angiogenesis are the ADAMs family of disintegrin-metalloproteases that have been implicated in modulating many cellular processes including adhesion, fusion, differentiation and surface protein shedding (10). ADAM's proteins were initially identified as important regulators of gamete fusion but have since been implicated in several other physiological processes including neurogenesis, myogenesis and the regulation of the inflammatory response (11, 12). The presence of a disintegrin domain has been shown to mediate integrin binding, although the physiological consequence of this activity in many ADAMs family members remains controversial. However, the biological function of their metalloprotease activity shows increasing prominence in the process of protein ectodomain shedding. For example, ADAM 17 or TACE has been shown to proteolytically process the precursor form of the pro-inflammatory cytokine TNFα, thereby promoting the release of the active cytokine from the cell surface (13). In addition, mammalian ADAM 10 (MADM) was also shown to possess TNFα-converting activity whereas the Drosophila ortholog KUZ is known to regulate notch signalling through cleavage of its extracellular domain promoting lateral inhibition during neurogenesis (14, 15) Furthermore, ADAM 13 which is expressed in Xenopus neural crest cells, is necessary for their migratory activity required for later stages of neurogenesis and this is thought to be due to the re-modelling (cleavage) of Fn by the metalloprotease domain (16, 17). Lastly, ADAM 17 has been reported to be responsible for the ectodomain shedding of GP1bα and GP V, components of the receptor complex for vWF, from platelets after treatment with aspirin (18). Therefore, it is conceivable that members of the ADAMs family could regulate VEGF A mediated responses through mechanisms involving these established biological activities or via hitherto unappreciated modes of action.
Previous studies have shown that ADAM 15 is expressed in cultured EC and smooth muscle cells (SMC) and its expression is elevated in diseased vascular tissue (19) suggesting a role in pathological vascular remodelling. ADAM 15 is a family member with a predicted active metalloprotease which is expressed in cells of haematopoeitic and neural origin. The human orthologue of ADAM 15, metargidin, is the only ADAM family member with an active canonical RGD sequence within its disintegrin domain (20). ADAM 15 has also been co-localised to the adherens junctions of endothelium with VE-cadherin suggesting that ADAM 15 may be involved in processes involving these cell junctions (21).
In 2003, it was shown that ADAM 15−/− null mice develop normally but exhibit impaired pathological angiogenesis. This lead to speculation about a potential role for ADAM 15 in pathological neovasculization in mice (22).
In 2004, Blobel et al. (51) suggested that therapeutic agents which inhibit ADAM 9 and/or ADAM 15 might be used for the treatment of vascularization-related disease or wound healing. Antibodies, small molecule therapeutics, antisense RNAs and an agent for introducing targeted mutations in the genetic sequence of ADAM 9 or ADAM 15 were suggested in this regard although not exemplified. Suitable targets for the development of antibody therapies were said to include intact ADAM9, intact ADAM15, portions of ADAM9 or ADAM15 derived from the extracellular portions of the protein; the protease and disintegrin domains of the extracellular portions were also postulated as potential targets. However, no data in support of the action of such therapeutic agents was presented and no antibodies were exemplified.
In 2005, Rahman et al. (52) disclosed two polyclonal rabbit sera (Ab 576 and Ab 577) against a peptide corresponding to amino acid residues 346-359 of the human ADAM 15 polypeptide. (Amino acid residues 346-359 fall within the metalloprotease domain of ADAM 15 proximal to the predicted catalytic cleft). Affinity-purified antibodies derived from these sera were used to investigate the effect that ADAM 15 has on endothelial cell migration in a Boyden chamber assay. In direct contrast to the suggestions put forward in Blobel (51), these anti-metalloprotease domain directed antibodies were found to promote a 2-3 fold elevation in endothelial cell migration (FIG. 5) of Rahman et al. This effect was confirmed by gene silencing (siRNA to ADAM 15) experiments. These experiments appeared to show that antibodies against the metalloprotease domain of ADAM 15 could therefore potentially promote vascularization and hence such antibodies would not be suitable as agents for the prevention of neovascularization or angiogenesis.